Protein tyrosine kinases (PTKs) and their substrates play a critical role in numerous cellular processes such as proliferation, differentiation, motility, and apoptosis. Aberrant kinase activation and the accompanying changes in the phosphotyrosine (designated also as pTyr or pY) signaling network are hallmarks of numerous cancers. A primary mechanism used by the cell to interpret pTyr-mediated signals relies on modular protein domains that bind specifically to tyrosine-phosphorylated proteins. The Src homology 2 (SH2) domain is the most prevalent of these modular domains, and plays a central role in PTK signaling pathways. Different pTyr sites recruit different SH2 domain-containing proteins, which in turn, activate different signaling pathways.
PTKs comprise, inter alia, receptor tyrosine kinases, including members of the epidermal growth factor kinase family. Enhanced activities of PTKs have been implicated in a variety of malignant and non-malignant proliferative diseases. In addition, PTKs are known to play a role in the regulation of cells of the immune system.
PTKs are important drug targets for cancer treatment. Current anti-cancer drugs are largely based on small-molecule kinase inhibitors or humanized antibodies. These drugs often display a broad specificity to a group of related kinases, and patients eventually develop resistance to the drugs after being on the treatment for a year or so.
An alternative idea of inhibiting PTK signaling is blockage of downstream signaling by masking phosphotyrosine of a PTK substrate. Although phosphotyrosine-specific antibodies have high affinity to pTyr-containing polypeptides, they cannot be used inside of cells.
The pTyr-specific antibody (U.S. Pat. No. 6,824,989) is widely used to detect pTyr contained in biological specimen. However, an antibody cannot be used inside of a living cell. An IgG antibody molecule is heterotetrameric protein with the total molecular weight of ˜150 kDa that is secreted to the extracellular space by B cells in the immune system. An antibody contains disulfide bonds, works outside of a cell in the immune system, and is not designed to function in cytoplasm or to penetrate the cell plasma membrane. Therefore, the pTyr-specific antibody cannot be used as an in vivo agent for interfering with intracellular signaling events involving protein tyrosine phosphorylation inside of living cells.
SH2 domain containing proteins work downstream of PTK signalling and are points of signal integration. An SH2 domain contains ˜100 amino acid and is approximately 15 times smaller than an antibody molecule. Isolated SH2 domains, when delivered or expressed in cells, can compete with endogenous signaling proteins that bind to pTyr sites. However, natural SH2 domains are designed to mediate transient interaction with their cognate binding sites to assure dynamic cellular signaling. In other words, a natural SH2 domain is inherently designed not to block PTK, signaling pathways in vivo. Because of this feature, a natural SH2 domain is not usable as a strong inhibitory reagent.
U.S. Pat. No. 5,786,454 (“U.S. 454”) discloses SH2 domains that possess an altered binding site that changes sequence recognition specificity. It has also been reported that modifications of the target-binding site of an SH2 domain, that include deletion, substitution, or introduction of unnatural amino acids, can change sequence recognition specificity of the SH2 domain (Songyang, et al. (1995) J. Biol. Chem., Vol. 270, pp. 26029; Kimber, et al. (2000) Mol. Cell, Vol. 5, pp. 1043; Kaneko, et al. (2010) Sci. Signal., Vol. 3, pp. ra34; Virdee et al. (2010) Chemistry & Biology, Vol. 17, pp. 274). SH2 variants created by this manner exhibit enhanced specificity for their cognate target polypeptides in some cases. However, these SH2 variants generally bind to their cognate target polypeptides with similar affinities as the corresponding natural SH2 domains.